Nested sets of protein fragments and their use in epitope mapping: characterization of the epitope for the S4D5 monoclonal antibody binding to receptor associated protein.
J Immunol Methods
; 180(1): 53-61, 1995 Mar 13.
Article
en En
| MEDLINE
| ID: mdl-7534803
The present report describes a new general procedure by which linear and some structure-dependent epitopes may be mapped in a protein antigen using a nested set of protein fragments prepared from partial proteolysis products of a recombinant protein. Briefly, the antigen, fused to an affinity tag, is partially fragmented and affinity sorted under denaturing conditions to produce a nested set of polypeptides, consisting of N- (or C-)terminal fragments. Immunoblots of SDS-PAGE fractionated sets of fragments are therefore directly readable in terms of molecular mass--i.e., approximate sequence positions--that identify sequence segments harbouring an epitope and any additional structural elements, required to maintain epitope conformation. Blots of N- and C-terminal nested sets of polypeptide fragments representing the human receptor associated protein (RAP) were prepared and probed with mAb S4D5 (Moestrup and Gliemann, 1991). Fragments 1-177 and 94-323 were the shortest fragments detected by the antibody, suggesting the presence of an epitope within the 94-177 segment. Independent mapping based on recombinant fragments of the RAP homologue, rat Heymann nephritis antigen, confirmed that the epitope resides in the Pro115-Asp177 segment. The model study demonstrates the utility of nested sets of protein fragments as fast and inexpensive tools for epitope mapping.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fragmentos de Péptidos
/
Mapeo Peptídico
/
Glicoproteínas de Membrana
/
Receptores Inmunológicos
/
Anticuerpos Monoclonales
/
Epítopos
Tipo de estudio:
Risk_factors_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
J Immunol Methods
Año:
1995
Tipo del documento:
Article
País de afiliación:
Dinamarca
Pais de publicación:
Países Bajos